ABCMdb

PMID: 23440202

Jih KY, Hwang TC
Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.
Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR`s responsiveness to ATP. Login to comment 3 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770`s effect on G551D-CFTR. Login to comment 6 ABCC7 p.Arg352Cys We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Login to comment 16 ABCC7 p.Gly551Asp For example, defects in NBD dimer formation have been proposed to account for the low open probability (Po) seen in ƊF508- and G551D-CFTR, the first and third most common CF-associated mutations (13-15). Login to comment 20 ABCC7 p.Gly551Asp Vx-770 has shown promising clinical outcomes for patients carrying the G551D mutation (19, 20) and was recently approved by the FDA. Login to comment 25 ABCC7 p.Gly551Asp Perhaps the most puzzling observation is that Vx-770 not only prolongs the open time of WT-CFTR, which is controlled by ATP hydrolysis; it also increases the activity of G551D-CFTR, a mutation that likely prevents ATP-induced NBD dimerization from happening due to its unique position in the signature sequence (14) and for the same reason eliminates ATP hydrolysis (22, 23). Login to comment 40 ABCC7 p.Arg352Cys 11 www.pnas.org/cgi/doi/10.1073/pnas.1215982110 finding of a mutant, R352C-CFTR, which allows quantification of ATP hydrolysis-triggered open-to-open transition, imparts a straightforward approach to examine how Vx-770 affects the reentry pathway. Login to comment 41 ABCC7 p.Arg352Cys Our data with R352C-CFTR not only support the hypothesis that Vx-770 lengthens the open time of WT-CFTR by increasing the frequency of reentry, but also spawn unique targets for CFTR potentiators that could complement the action of Vx-770, a crucial step toward the ultimate goal of curing CF. Login to comment 44 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because ATP-induced NBD dimerization and subsequent ATP hydrolysis are likely abolished by the G551D mutation, these two plausible mechanisms may not easily account for the effect of Vx-770 on G551D-CFTR. Login to comment 47 ABCC7 p.Gly551Asp If Vx-770 shifts the equilibrium of the C2 ࢒ O2 transition (i.e., ATP-independent gating) toward the O2 state, one expects to observe an increased activity of G551D-CFTR as ATP-independent openings are preserved in this mutant, as well as for WT-CFTR because more reentry events will occur with a stabilized O2 state. Login to comment 59 ABCC7 p.Gly551Asp Similarly, we found that Vx-770 increases the mean macroscopic current of G551D-CFTR by 12.8 &#b1; 1.9 fold (n = 12; Fig. S1). Login to comment 60 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Taking account of the basal Po of G551D-CFTR (~120 times lower than WT) (14), we estimated a Po of ~0.04 for Vx-770- treated G551D-CFTR. Login to comment 80 ABCC7 p.Gly551Asp *P < 0.05. those reported previously (an eightfold increase by Vx-770 with a rectified Po of ~0.15) (18), and thus demand further mechanistic studies of Vx-770 on G551D. Login to comment 105 ABCC7 p.Arg352Cys Effects of Vx-770 on R352C-CFTR. Login to comment 109 ABCC7 p.Arg352Cys Fortunately, our recent discovery of a CFTR mutant, R352C, that exhibits different single-channel conductance for the O1 and O2 states grants us a unique opportunity to directly assess the effects of Vx-770 on the lifetime of the O2 state. Login to comment 122 ABCC7 p.Arg352Cys report (24), most of the opening bursts in R352C-CFTR contain one O1 ࢐ O2 transition; only ~10% of the openings are categorized as events with more than one O1 ࢐ O2 transition. Login to comment 131 ABCC7 p.Gly551Asp As a CFTR potentiator, Vx-770 is unique in that it increases the open time of WT-CFTR, which is mainly controlled by ATP hydrolysis (11, 28, 31, 32) and the activity of G551D-CFTR, a mutant that does not respond to ATP (14). Login to comment 132 ABCC7 p.Gly551Asp Indeed, the classical methods used to lengthen the open time of WT-CFTR by abolishing ATP hydrolysis with either mutations or nonhydrolyzable ATP analogs fail to work on the G551D channels (14, 37). Login to comment 133 ABCC7 p.Gly551Asp One possible explanation for this apparent dichotomy is that Vx-770 works on WTand G551D-CFTR channels via two unrelated mechanisms. Login to comment 134 ABCC7 p.Gly551Asp However, the observation that the magnitude of Vx-770`s effects on ATP-independent activity of WT-CFTR (~9&#d7;, Fig. 2D) is similar to the fold increase of G551D-CFTR (~8&#d7;, ref. 18, ~13&#d7; in the current study) suggests a unified theory may explain Vx-770`s dual actions. Login to comment 138 ABCC7 p.Gly551Asp Based on this model, one can explain Vx-770`s effects on both WTand G551D-CFTR by proposing that the drug shifts the equilibrium of C2 ࢒ O2 transition toward the O2 state. Login to comment 141 ABCC7 p.Arg352Cys Furthermore, by examining the effects of Vx-770 on R352C-CFTR, we were able to show that this compound indeed increases the frequency of reentry events (Fig. 4 and Table 1). Login to comment 142 ABCC7 p.Arg352Cys Notably, the reentry pathway was first suggested by a report using PPi as a tool to fish out a unique posthydrolytic state (36) and later established by scrutinizing the R352C mutant channel that exhibits hydrolysis-dependent open channel conductance: unequal single-channel current amplitudes between pre-and posthydrolytic states (24). Login to comment 153 ABCC7 p.Arg352Cys Effects of Vx-770 on R352C-CFTR. Login to comment 154 ABCC7 p.Arg352Cys (A and B) Representative single-channel traces for R352C-CFTR in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment 157 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Summary of the effects of Vx-770 on the gating patterns exhibited in R352C-CFTR and W401F/R352C-CFTR Total Experimental condition Without Vx-770* R352C (%) 834 (55) 301 (20) 173 (11) 39 (3) 169 (11) 1,516 (100) R352C/W401F (%) 733 (44) 326 (19) 122 (7) 28 (2) 474 (28) 1,683 (100) With 200 nM Vx-770 R352C (%) 578 (57) 126 (12) 73 (7) 20 (2) 217 (21) 1,014 (100) R352C/W401F (%) 411 (37) 162 (15) 79 (7) 31 (3) 425 (38) 1,108 (100) Five different categories of the gating pattern are illustrated. Login to comment 161 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe *Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ࢒ O2) primarily affected by Vx-770 are supposed to take place in CFTR`s TMDs (24). Login to comment 171 ABCC7 p.Gly551Asp Whereas the exact reason for the difference between current results and those reported by Eckford et al. (39) awaits further studies, it is important to point out that an accurate estimation of the Po for G551D-CFTR from microscopic kinetic analysis is challenging because the number of channels cannot be reliably determined. Login to comment 172 ABCC7 p.Gly551Asp Although Vx-770 is now an established treatment for patients with CF, its clinical impacts are somewhat limited as the G551D mutation only accounts for ~3% of CF-associated mutations (www. Login to comment 174 ABCC7 p.Gly551Asp Furthermore, the sweat chloride level, a parameter directly reflecting CFTR activity in vivo and thus a widely used diagnostic tool for CF, in patients taking Vx-770 (19, 20) were still higher than those found in patients with mild-form CF (19, 20, 40), suggesting that Vx-770 alone is insufficient to completely rectify the dysfunction associated with the G551D mutation in vivo. Login to comment 178 ABCC7 p.Trp401Phe Lately, we discovered that W401F, a conserved mutation in NBD1, increases the reentry frequency without significantly altering the O2 ࢐ C2 transition (24). Login to comment 179 ABCC7 p.Trp401Phe Although how the W401F mutation modifies the function of NBDs is unclear, this finding does raise the possibility that manipulating NBD function can complement the action of Vx-770. Login to comment 180 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe As shown in Fig. S3 and Table 1, the W401F mutation almost doubles the reentry frequency of R352C-CFTR and also significantly enhances the effect of Vx-770 (Fig. 5, Table 1 and Fig. S3). Login to comment 207 ABCC7 p.Arg352Cys Because the single-channel conductance is reduced by the R352C mutation, a pipette solution with 375 mM Cl-was used (in millimoles): Fig. 5. Login to comment 208 ABCC7 p.Trp401Phe W401F mutation and Vx-770 work additively on the reentry pathway. Login to comment 209 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment 210 ABCC7 p.Arg352Cys Frequency of opening bursts containing multiple rounds of O1 ࢐ O2 transition (summarized in Table 1) is increased and the overall open time is prolonged (Fig. S3D) compared with R352C-CFTR recorded in the same condition. Login to comment 213 ABCC7 p.Arg352Cys For inside-out configuration, the 150 mM Cl- perfusion solution contained (in millimoles): 150 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG), the 375 mM Cl- perfusion solution used for R352C-CFTR contained (in millimoles): 375 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG). Login to comment 223 ABCC7 p.Arg352Cys For kinetic analysis of R352C-CFTR, three current levels (C, O1, and O2) were determined from all point histograms. Login to comment 271 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 14. Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 286 ABCC7 p.Gly551Asp Accurso FJ, et al. (2010) Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N Engl J Med 363(21):1991-2003. Login to comment 288 ABCC7 p.Gly551Asp Ramsey BW, et al.; VX08-770-102 Study Group (2011) A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med 365(18):1663-1672. Login to comment 334 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Cai Z, Taddei A, Sheppard DN (2006) Differential sensitivity of the cystic fibrosis (CF)- associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment